JP2012521364A - How to adjust circadian rhythm - Google Patents

Abstract

The present disclosure described the role of AMPK in circadian rhythms and methods of screening for agents that modulate such rhythms, compositions useful for modulating such rhythms, and uses thereof. The present disclosure also provides a composition comprising an AMPK agonist formulated in combination with a second active ingredient that alters circadian rhythm. In one embodiment, the second active ingredient is a sleep aid. In further embodiments, the composition is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial delivery or subcutaneous injection.

Description

(Cross-reference to related applications)
This application claims the benefit of US Provisional Patent Application No. 61 / 162,219, filed Mar. 20, 2009, which is incorporated herein by reference.

(Authorization of government support)
This study was supported by National Institutes of Health grant numbers DK057978, DK062434, CA104838, DK080425, and EY016807. The United States government has certain rights in this invention.

(Field of Invention)
The present disclosure relates to the use of agonists and antagonists of AMP-activated protein kinase (AMPK) to modulate circadian rhythm. More specifically, the present disclosure provides compositions and methods for screening and modulating sleep behavior.

  The circadian clock harmonizes behavioral and physiological processes to the daily light-dark cycle by driving the rhythmic transcription of thousands of genes in mammalian tissues.

  The present disclosure provides methods and compositions for altering circadian rhythm in a mammalian subject such as a human. The present disclosure demonstrates that AMPK is modified in the brain and other tissues in the body during the circadian cycle of a mammalian subject. In one embodiment, the present disclosure provides the use of an AMP kinase agonist or antagonist for the manufacture of a medicament for modulating circadian rhythm in a subject. In one embodiment, the AMPK agonist is AICAR. In another embodiment, the AMPK antagonist is an antibody or compound C or analog or derivative thereof. In yet another embodiment, the AMPK agonist comprises a formulation or derivatization that can cross the blood brain barrier. In still further embodiments, the AMPK agonist is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial or subcutaneous injection.

  The present disclosure also provides a composition comprising an AMPK agonist formulated in combination with a second active ingredient that alters circadian rhythm. In one embodiment, the second active ingredient is a sleep aid. In further embodiments, the composition is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial delivery or subcutaneous injection.

  The present disclosure provides a method of modulating sleep in a mammal, comprising administering to the mammal an amount of an AMPK agonist or antagonist effective to modulate circadian rhythm in the mammal.

  The present disclosure is a method for identifying an agent that modulates circadian rhythm or sleep in a subject, comprising: (a) contacting a sample comprising an AMPK pathway with at least one test agent; and (b) Comparing the activity of AMPK or AMPK pathway in the presence and absence of a test agent, wherein the test agent that alters the activity indicates an agent having circadian rhythm regulating activity And a method comprising:

  The above and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

1A-D show that disruption of AMPK signaling changes circadian rhythm in MEF. (A) Unsynchronized paired wild type (AMPK ^{+ / +} ) or ampkα1 ^{− / −} ; ampkα2 ^{− / −} (AMPK − / −) mouse embryonic fibroblasts are exposed to 50% horse serum for 2 hours. And then transferred to a medium containing 25 mM glucose supplemented with 25 mM glucose, 0.5 mM glucose or 1 mM AICAR. Quantitative PCR analysis was performed using cDNA samples collected at the indicated times after stimulation. Data represent the mean of two independent experiments each analyzed in triplicate. (B) Fibroblasts stably expressing Bmal1-luciferase were cultured in media containing the indicated amount of glucose with or without 2 mM AICAR. Shown are representative results of continuous monitoring of luciferase activity. (C and D) Quantification of circadian period (C) and amplitude (D) of Bmal1-driven luciferase activity from experiments performed as described in (B). Data in (C) and (D) represent mean ± standard deviation for 4 samples per condition. ANOVA analysis showed significant differences between categories. ^** P <0.01 for sample cultured with 25 mM glucose in Scheffe's post hoc analysis. 2A-C show that AMPK activity and nuclear localization are subject to circadian regulation. (A) Immunoblotting for phospho-Raptor-S792 (pRaptor), Raptor, phospho-ACC1-S79 (pACC1) and ACC1 was performed on whole cell lysates prepared from mouse liver collected at circadian times as described. It was. The blot is representative of 3 independent experiments. (B) Quantitative PCR analysis of cDNA prepared from mouse liver collected at circadian time as described. Each data point represents the mean ± standard deviation of three samples collected from each unique animal and analyzed in quadruplicate. (C) Nuclear extracts were prepared from the livers of 2 mice at each of the circadian times described. Protein levels of AMPKα1, AMPKα2, PER2, CRY1 and REVERBα were analyzed by immunoblotting. Paired wild type (α1 + / +) and ampkα1 ^{− / −} (α1 − / −) or wild type (α2 + / +) and ampkα2 ^{− / −} (α2 − / −) mice collected at the circadian time described A nuclear extract from was used as a control for antibody specificity. 3A-C show that AMPK activation alters CRY stability and circadian rhythm in mouse liver. (A) Mice were injected with physiological saline or 500 mg AICAR per kg body weight, and liver samples were collected at 1 hour after Zeitgeber time (ZT, time after lighting) 6 or ZT18. Endogenous CRY1 was detected by immunoblotting in liver nuclear extracts. n. s. Indicates non-specific bands for assessing sample loading. Samples collected from wild type (CRY + / +) and cry1 ^{− / −} ; cry2 ^{− / −} (CRY ^{− / −} ) mice were used as controls for antibody specificity. Data represent representative results from two independent experiments. (B) LKB1 ^{+ / +} and LKB1 ^{fl / fl} mice were injected with adenovirus expressing Cre recombinase (Ad-Cre) via the tail vein. One to two weeks after Ad-Cre injection, mice were transferred to a stationary dark place and livers were collected at the circadian times described. CRY1, PER2 and REVERBα were detected by immunoblotting. (C) cDNA samples prepared from the liver described in (B) were analyzed by quantitative PCR analysis of dbp, reverbα, cry1 and per2 expression. All transcripts were normalized to u36b4 as an internal control. Each data point represents the mean ± standard deviation of three samples analyzed in quadruplicate. 4A-B show that the destruction of AMPK changes the circadian rhythm in MEF. 3T3 immortalized mouse embryonic fibroblasts (A) or paired wild type (AMPK ^{+ / +} ) or ampkα1 ^{− / −} ; ampkα2 ^{− / −} (AMPK ^{− / −} ) fibroblasts (B), 50% horse Stimulate by exposure to serum for 2 hours, then transfer to medium containing 25 mM glucose (black symbol), 0.5 mM glucose (grey symbol) or 25 mM glucose (red symbol) supplemented with 1 mM AICAR. did. Quantitative PCR analysis was performed using cDNA samples prepared from lysates collected at the indicated times after stimulation. Data represent the mean ± standard deviation of 2 or 3 independent experiments each analyzed in triplicate.

  Unless otherwise stated herein, the definitions of terms used are standard definitions used in the pharmaceutical arts. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes a mixture of two or more such carriers, and the like.

  Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes”, and “included” are interchangeable. , Not intended to be limiting.

  Where the description of the various embodiments uses the term “comprising”, in some specific cases, embodiments may be described instead using the words “consisting essentially of” or “consisting of”. It will be further understood by those skilled in the art.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods and materials are now described.

  All publications mentioned herein are hereby incorporated by reference in their entirety for the purpose of describing and disclosing the methods described in that publication that may be used in connection with the description herein. Incorporated herein. Publications throughout the text discussed above are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

  Circadian rhythms include, but are not limited to, the adjustment of sleep cycles, energy adjustments related to exercise and calorie reduction, and appropriate timing of physiological processes, endocrine and behavioral processes such as feeding / nourishment behavior Optimize biological efficiency by harmonizing The circadian rhythm is considered to contain at least three elements: (a) input path (s) that relay environmental information to the circadian pacemaker (clock); (b) circadian pacemaker that creates oscillation; And (c) output path (s) through which the pacemaker adjusts various output rhythms.

  The mammalian hypothalamic suprachiasmatic nucleus (SCN) acts as the primary pacemaker that aligns behavioral and physiological rhythms with the light-dark cycle. Initially, SCN was considered the only site of a self-supporting molecular pacemaker in mammals, but several papers have since shown that such molecular clocks are nearly ubiquitous. Unlike the SCN clock, the circadian clock in light-insensitive peripheral organs is tuned by the daily rhythm of feeding, allowing peripheral tissues to anticipate daily food consumption and optimize the timing of metabolic processes. Make it possible theoretically. Several papers support a role for the mammalian circadian clock in the regulation of transcription and metabolic physiology of important metabolic enzymes.

  As used herein, the term “circadian rhythm” is intended to mean a regular variation in physiological and behavioral parameters that occurs over the course of about 24 hours. Such activities include sleep cycles and nutritional cycles. In one embodiment, the circadian rhythm may include energy adjustments associated with exercise and calorie reduction. For example, the methods and compositions of the present disclosure can be used to regulate energy use and sleep in the body. As described below, AMPK agonists induce a metabolic shift to the production of ATP by lipid catabolism and, at the same time, reduce ATP use by resting the body. Thus, AMPK agonists can induce both exercise catabolism / metabolic processes as well as rest / sleep states.

  As used herein, the term “modulate” when used with respect to circadian rhythms refers to altering physiological functions, endocrine functions or behaviors regulated by an animal's circadian timing system, or circadian rhythms. It is intended to mean altering cell function that exhibits gender. Exemplary physiological functions regulated by the animal's circadian timing system include body temperature, autonomic regulation, metabolism and sleep-wake cycle. Exemplary metabolic functions include weight gain including weight gain or loss and body fat percentage gain or loss, as well as weight loss control. Exemplary endocrine functions regulated by the animal circadian timing system are pineal melatonin secretion, ACTH cortisol secretion, thyroid stimulating hormone secretion, growth hormone secretion, neuropeptide Y secretion, serotonin secretion, insulin-like growth factor I Includes type secretion, corticotropin secretion, prolactin secretion, gamma-aminobutyric acid secretion and catecholamine secretion. Exemplary behaviors regulated by the animal circadian timing system include exercise (motor rhythm), mental agility, memory, sensorimotor integration, feeding, REM sleep, NREM sleep and emotion.

  AMP-activated protein kinase (AMPK) is recognized as a central mediator of metabolic signals that are well conserved throughout phylogeny. AMPK is a heterotrimeric protein kinase that contains a catalytic (α) subunit and two regulatory (β, γ) subunits. It is activated upon phosphorylation by LKB1 in the presence of a high AMP / ATP ratio or by CAMKKβ in the presence of elevated intracellular calcium. Biochemical and bioinformatics studies have established optimal amino acid sequence relationships that are likely to be phosphorylated by AMPK.

  AMP-activated protein kinase (AMPK) and AMPK kinase (AMPKK) are associated with the protein kinase cascade. The AMPK cascade regulates fuel production and utilization intracellularly. For example, AMPK activity increases with low cellular fuel (eg, increased AMP concentration). Once activated, AMPK functions to store ATP or facilitate alternative methods of ATP generation.

  AMPK is expressed in several tissues including liver, brain and skeletal muscle. Activation of AMPK activates liver fatty acid oxidation and ketone body formation, inhibits cholesterol synthesis, adipogenesis and triglyceride synthesis, inhibits adipocyte lipolysis and adipogenesis, skeletal muscle fatty acid oxidation and muscle glucose It has been shown to stimulate uptake and regulate insulin secretion by pancreatic beta cells.

  Activation of AMPK can be induced by increasing the concentration of AMP. The γ subunit of AMPK undergoes a conformational change so as to expose the active site (Thr-172) on the α subunit. A conformational change of the γ subunit of AMPK can be achieved when the concentration of AMP is increased. By increasing the concentration of AMP, the conformational change of the γK subunit of AMPK occurs when two AMPs bind to the two Bateman domains in this subunit. This role of AMP has been demonstrated in experiments showing AMPK activation by 5-amino-4-imidazolecarboxamide ribotide (ZMP), an AMP analog derived from 5-amino-4-imidazolecarboxamide riboside (AICAR). The Similarly, antagonists of AMP include the use of inhibitory antibodies that inhibit activation of downstream kinases by AMPK.

  Sleep deprivation (SD) increases neural activity. Sustained neuronal activity decreases cellular energy sufficiency (AMP levels increase and ATP decreases). This in turn causes a change in the cellular energy sensor AMPK. As discussed above, AMPK regulates various kinase cascades.

  CLOCK and BMAL1 are polypeptides that induce transcription of genes associated with circadian rhythm by formation of heterodimers. During a typical circadian cycle, the molecular mechanism fluctuates between two cycles that form an internal clock with two interconnected transcription / translation feedback loops. The positive arm of the feedback loop is driven by the basic helix-loop-helix-PAS (Per-Arnt-Sim) domain-containing transcription factors CLOCK and BMAL1. The CLOCK / BMAL1 heterodimer activates transcription of the clock genes cryptochrome (Cry1 and Cry2), period (Per1 and Per2), and Rev-Erbα. PER and CRY proteins move to the nucleus where they interact with CLOCK / BMAL1 to down regulate transcription and generate a negative arm of the main feedback loop.

  Post-translational modifications of clock proteins (eg, phosphorylation and dephosphorylation) determine protein localization, intermolecular interactions and stability, and thus regulate the circadian clock cycle. The present disclosure demonstrates that this post-translational regulation can be modulated by AMPK activity and thus AMPK agonists and antagonists can have a role in the regulation of the circadian clock.

  The present disclosure relates to compounds that bind to AMP-activated protein kinase (AMPK) or otherwise activate or inactivate it (some of these) to affect sleep or other circadian processes Provides the use of currently used for the treatment of diabetes. The present disclosure demonstrates that genetic or pharmacological manipulation of AMP-activated protein kinase activity alters circadian rhythms in cultured cells and intact animal livers. The present disclosure also demonstrates that AMP kinase is expressed in the suprachiasmatic nucleus (SCN), the location of the so-called “major pacemaker” that governs the timing of sleep-wake cycles and other physiological rhythms. Currently available therapies do not cross the blood brain barrier and are therefore not useful for the regulation of sleep disorders.

  Regulation of circadian rhythm by AMPK is useful for the treatment of sleep disorders including but not limited to insomnia, as AMPK modulators that cross the blood brain barrier regulate downstream kinase activity associated with circadian rhythm It is suggested. Furthermore, certain circadian polypeptides, including but not limited to CLOCK, BMAL1, PER and CRY-1 and -2, are regulated by phosphorylation and dephosphorylation and are present in tissues outside the brain. Thus, modulating AMPK activity in non-neural tissues may be important for setting circadian rhythms by the kinase cascade and ultimately regulating phosphorylation and dephosphorylation of downstream polypeptides.

  Several pharmacological agents that activate AMPK are currently in clinical use for the treatment of diabetes and are being clinically tested for several types of cancer.

  AMP kinase agonists such as AICAR have been studied for insulin regulation, diabetes and obesity. However, AMP kinase has not been previously demonstrated to regulate circadian rhythm and sleep behavior. The present disclosure demonstrates that modulating AMPK activity can have effects on downstream processes including post-translational modifications of proteins associated with circadian rhythm. In one embodiment, the present disclosure shows that AMPK agonists and antagonists can be used to modulate circadian rhythm in a subject. For example, AMPK is demonstrated by the present disclosure to play a role in regulating transcription that activates heterodimer CLOCK / BMAL1.

  Various AMPK agonists are known in the art. Methods and compositions comprising such AMPK agonists are provided herein. The use of such AMPK agonists can provide a way to adjust circadian rhythm. In one embodiment, the AMPK agonist comprises an AICAR compound. Other compounds useful in the disclosed methods include biguanide derivatives, analogs of AICAR (eg, those disclosed in US Pat. No. 5,777,100, incorporated herein by reference) and AICAR bio Including prodrugs or precursors of AICAR that increase availability, such as those disclosed in US Pat. No. 5,082,829, incorporated herein by reference, all known to those skilled in the art is there. Other activators of AMPK include those described in US Patent Application Publication No. 20060287356 to Iyengar et al., The disclosure of which is incorporated herein by reference. Conventionally known AMPK activating compounds include, for example, leptin, adiponectin and metformin, AICAR (5-aminoimidazole-4-carboxamide). Other AMPK agonists include, but are not limited to, phenformin, ZMP, DRL-16536 (Dr. Reddy's / Perlecan Pharma), BG800 compound (Betagenon), furan-2-carboxylic acid derivative (Hanall, KR; International See also published application WO / 2008/016278 (also incorporated herein by reference), A-769662 (Abbott) (Structure I; Cool et al., Cell Metabol. 3: 403-416, 2006). See also year); AMPK agonists under development by Metabasis as described in International Publication No. WO / 2006/033709; MT-39 series of compounds (Mercury Therapeutics); and Trans By ech Pharma including the AMPK agonists under development.

例えば、ＡＩＣＡＲは、細胞に取り込まれ、ＡＭＰＫを活性化することが示されているＡＭＰ類似体であるＺＭＰに変換される。ＺＭＰは、細胞内ＡＭＰ模倣物として作用し、十分に高いレベルまで蓄積されると、ＡＭＰＫ活性を刺激できる（Ｃｏｒｔｏｎ， Ｊ． Ｍ．ら、Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．、２２９巻：５５８頁（１９９５年））。しかし、ＺＭＰは、他の酵素の調節においてもＡＭＰ模倣物として作用し、よって、特異的ＡＭＰＫ活性化因子ではない（Ｍｕｓｉ， Ｎ．およびＧｏｏｄｙｅａｒ， Ｌ． Ｊ． Ｃｕｒｒｅｎｔ Ｄｒｕｇ Ｔａｒｇｅｔｓ−−Ｉｍｍｕｎｅ， Ｅｎｄｏｃｒｉｎｅ ａｎｄ Ｍｅｔａｂｏｌｉｃ Ｄｉｓｏｒｄｅｒｓ ２巻：１１９頁（２００２年））。

For example, AICAR is taken up by cells and converted to ZMP, an AMP analog that has been shown to activate AMPK. ZMP acts as an intracellular AMP mimetic and, when accumulated to a sufficiently high level, can stimulate AMPK activity (Corton, JM et al., Eur. J. Biochem., 229: 558 (1995). Year)). However, ZMP also acts as an AMP mimetic in the regulation of other enzymes and is therefore not a specific AMPK activator (Musi, N. and Goodyear, L. J. Current Drug Targets--Immune, Endocrine and Metabolic Disorders 2: 119 (2002)).

  The present disclosure provides a method of stimulating a specific cycle of the circadian clock in a subject by using either an AMPK agonist or an AMPK antagonist. In one embodiment, an AMPK agonist is used to promote the circadian cycle associated with increased CLOCK / BMAL1 transcriptional activity. In one embodiment, AMPK agonists promote sleep effects through energy conservation signaling through corresponding kinase cascades. The method includes administering to the subject an amount of an AMPK agonist sufficient to stimulate an energy deficiency condition in the subject. A state in which the γ subunit of AMPK undergoes a conformational change due to an “energy deficient state”. Promoting sleep effects means that such effects are improved in a subject more than occurs in the absence of an AMPK agonist.

  The disclosed method induces the desired circadian cycle status in a subject receiving the AMPK agonist alone, or including the formulation, including, but not limited to, administration methods, dosages and formulations well known to those of ordinary skill in the pharmaceutical arts. Any administration method, dosage, and / or use of the formulation in combination with other circadian regulators or sleep aids having the desired results is envisioned.

  An AMPK agonist of the present disclosure may be administered to a human or animal in the form of a drug. Alternatively, AMPK agonists may be incorporated into a variety of foods and beverages or pet foods for consumption by humans or animals. An AMPK agonist may be used in a general food or beverage, or a functional food or beverage, a food for a subject suffering from a disease, or a food for a particular health use (this food (or The beverage) has a label stating that it has physiological functions); for example, it may be used as a sleep aid.

  The AMPK agonist alone or in combination with other sleep aids or active ingredients may be formulated into an oral solid product such as a drug, eg, a tablet or granule, or an oral liquid product such as a solution or syrup.

  The mode of administration of the AMPK agonist or formulation in the disclosed methods is not limited thereto, but is intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal , Including epidural, intradural, intracranial, intraventricular and oral routes. In a specific example, the AMPK agonist is administered orally. Other convenient routes for administration of AMPK agonists include, for example, infusion or bolus administration, absorption through topical, epithelial or dermal mucosal layers (such as oral mucosa, rectum and intestinal mucosa), ocular, nasal and transdermal. Including. Administration can be systemic or local. Pulmonary administration (eg, by inhaler or nebulizer) can also be used, eg, by using a formulation containing an aerosol.

  As described in more detail below, the AMPK agonist can be administered by oral, parenteral, intramuscular, intravascular, or other suitable route. In one embodiment, the AMPK agonist is administered epidurally. In one embodiment, the AMPK agonist is formulated to facilitate passage through the blood brain barrier.

  In certain embodiments, it may be desirable to administer the AMPK agonist locally. This may involve, for example, topical or regional infusion or perfusion, topical application (eg wound dressings), injection, catheters, suppositories or implants (eg, sialastic membranes or fibers). Including, for example, implants formed from porous, non-porous or gelatinous materials.

  In addition to the use of agonists, it should be appreciated that antagonists can also be used to stimulate wakefulness. The present disclosure also provides a method of promoting an active state comprising administering an agent that antagonizes AMPK activity, thereby setting metabolism and activity for a “wake” or “activity” cycle. In one embodiment, the AMPK antagonist is an inhibitory antibody. In one embodiment, the AMPK antagonist is a small molecule inhibitor (eg, Compound C (Dorsomorphin, 6- [4- (2-piperidin-1-yl-ethoxy) -phenyl)]-3-pyridine-4 -Yl-pyrazolo [1,5-a] -pyrimidine), its analogues, derivatives or salts.

  In other embodiments, a pump (eg, an implanted minipump) may be used to deliver an AMPK agonist or formulation (eg, Langer Science 249, 1527, 1990; Safeton Crit. Rev. Biomed. Eng. 14). Vol. 201, 1987; Buchwald et al., Surgery 88, 507, 1980; Saudek et al., N. Engl. J. Med. 321, 574, 1989). In another embodiment, the AMPK agonist or formulation is delivered in vesicles, particularly liposomes (eg, Langer, Science 249, 1527, 1990; Liposomes in the Disease of Infectious Disease and Cancer, et al., Lopez-Berstein and Fiddler (eds.), Liss, NY, pages 353-365, 1989).

  In yet another embodiment, the AMPK agonist can be delivered in a controlled release formulation. Controlled release systems such as those discussed in the review by Langer (Science 249, 1527, 1990) are known. Similarly, polymeric materials useful in controlled release formulations are known (eg, Ranger et al., Macromol. ScL Rev. Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190, 1985). During et al., Ann. Neurol. 25, 351, 1989; see Howard et al., J. Neurosurg. 71, 105, 1989). For example, agonists include polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogel crosslinks or parents It may be combined with a class of biodegradable polymers useful for achieving controlled release of compounds, including amphiphilic block copolymers.

  The disclosed method contemplates the use of any dosage form of AMPK agonist or formulation thereof that delivers the agonist (s) to achieve the desired result. Dosage forms are generally known, for example Allen et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th edition, Philadelphia, PA: Lippincott Williams, page 7 Is taught in the book. Dosage forms for use in the disclosed methods include, but are not limited to, solid dosage forms and solid modified release drug delivery systems (eg, powders and granules, capsules and / or tablets); semisolid dosage forms and transdermal Systems (eg ointments, creams and / or gels); transdermal drug delivery systems; pharmaceutical inserts (eg suppositories and / or inserts); liquid dosage forms (eg solutions and dispersions); Or including sterile dosage forms and delivery systems (eg, parenterals and / or biologics). Specific exemplary dosage forms include aerosols (including metered doses, powders, solutions and / or no propellants); beads; capsules (conventional, controlled delivery, controlled release, enteric coating and / or Caplet; Concentrate; Cream; Crystal; Disc (including sustained release); Dropper; Elixir; Emulsion; Foam; Gel (including jelly and / or controlled release); Gum; implant; inhalation drug; injection; insert (including extended release); liposome; liquid (including controlled release); lotion; lozenge; metered spray (eg pump); mist; Nebulization solution; ophthalmic system; oil; ointment; vaginal suppository (oval); powder (packet, effervescent, suspension Turbid powder, suspension sustained release powder and / or solution powder); pellets; paste; solution (including long acting and / or reconstituted); strip; suppository (including sustained release) Suspensions (including lente, ultra lente, reconstituted); syrups (including sustained release); tablets (chewing, sublingual, sustained release, controlled release, delayed action, delayed release) Enteric coating, foaming agent, film coating, immediate dissolution, slow release); transdermal system; tincture; and / or cachet. Typically, dosage forms comprise an effective amount (eg, a therapeutically effective amount) of at least one pharmaceutically active ingredient that includes an AMPK agonist, and pharmaceutically acceptable excipients and / or other ingredients (eg, one And other active ingredients). The purpose of drug formulation is to appropriately administer the active ingredient (eg, AMPK agonist or AMPK antagonist) to the subject. The formulation is suitable for the mode of administration. The term “pharmaceutically acceptable” refers to use approved by a federal or state regulatory agency or used in the United States Pharmacopeia or other commonly recognized animals and more specifically in humans. Means that it is listed in the Pharmacopeia. Excipients for use in exemplary formulations include, for example, one or more of the following: binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, colorants. Preservatives, diluents, adjuvants and / or vehicles. In some cases, the excipients together constitute about 5% to 95% of the total weight (and / or volume) of a particular dosage form.

  The pharmaceutical excipients can be sterile liquids such as, for example, water and / or petroleum including peanut oil, soybean oil, mineral oil, sesame oil, animal oils, vegetable oils or oils of synthetic origin. Water is an exemplary carrier when the formulation is administered intravenously. Saline, plasma media, aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Oral formulations can include, but are not limited to, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. A more complete description of parenteral pharmaceutical excipients can be found in Remington, The Science and Practice of Pharmacy, 19th edition, Philadelphia, PA: Lippincott Williams & Wilkins, 1995, Chapter 95. Excipients include, for example, pharmaceutically acceptable salts for adjusting osmotic pressure, lipid carriers such as cyclodextrin, proteins such as serum albumin, hydrophilic agents such as methylcellulose, surfactants, Buffering agents, preservatives and the like may also be included. Other examples of pharmaceutical excipients are starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, Including propylene, glycol (propylene, glycol), water, ethanol and the like. If desired, the formulation can also contain minor amounts of wetting or emulsifying agents or pH buffering agents.

  In some embodiments involving oral administration, the oral dosage of an AMPK agonist is usually about 0.001 mg / kg body weight per day (mg / kg / day) to about 100 mg / kg / day, such as about 0.00. It is in the range of 01-10 mg / kg / day (unless stated otherwise, the amount of active ingredient is based on neutral molecules, which can be free acid or free base). For example, an 80 kg subject receives about 0.08 mg / day to 8 g / day, for example about 0.8 mg / day to 800 mg / day. A suitably prepared medicament for once daily administration thus contains 0.08 mg to 8 g, for example 0.8 mg to 800 mg. In some cases, formulations containing AMPK agonists or antagonists may be administered in divided doses twice, three times, or four times daily. For twice daily administration, a suitably prepared medicament as described above contains 0.04 mg to 4 g, for example 0.4 mg to 400 mg. Dosages outside the above ranges may be required. Examples of daily doses that can be given in the range of 0.08 mg to 8 g per day are 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, Contains 400 mg, 500 mg, 600 mg, 800 mg, 1 g, 2 g, 4 g and 8 g. These amounts can be divided into smaller doses if administered more than once a day (eg, half the amount at each administration if the drug is taken twice a day).

  In some method embodiments, including administration by injection (eg, intravenous or subcutaneous injection), the subject receives an injection dose that delivers the active ingredient in approximately the amounts described above. The amount may be adjusted to account for differences in delivery efficiency due to injectable drug forms that avoid the digestive system. Such an amount can be obtained in several suitable ways, such as a low concentration and high volume of active ingredient in one long period or several times a day, a short period, eg once a day. High concentrations and low volumes of active ingredients may be administered. Conventional intravenous containing a concentration of active ingredient typically between about 0.01 and 1.0 mg / ml, such as 0.1 mg / ml, 0.3 mg / ml or 0.6 mg / ml The formulation may be prepared and administered in a daily dose equal to the daily dose described above. For example, an 80 kg subject who receives 8 ml of an intravenous formulation having a concentration of 0.5 mg / ml of active ingredient twice a day will receive 8 mg of active ingredient per day.

  In other embodiments, the AMPK agonist or antagonist (or formulation thereof) can be administered at about the same dose, incremental dose regimen, or loading dose regimen (eg, the loading dose is about 2-5 times the maintenance dose) throughout the treatment period. . In some embodiments, the dosage will vary during the period of use based on the condition of the subject receiving the composition, the apparent response to the composition, and / or other factors determined by one skilled in the art. In some embodiments, long term administration of an AMPK agonist or antagonist is contemplated, eg, to manage chronic insomnia or sleep-wake cycle disorders.

  The present disclosure also provides a method of screening for agents that modulate circadian rhythm by measuring AMPK activation or inhibition. A method for screening a compound that modulates circadian rhythm is provided by providing a cell, tissue or subject (eg, animal) comprising an AMPK pathway, and said subject is presumed to have circadian rhythm modulating activity. Contacting with an active agent, and measuring an effect on AMPK activity directly or by downstream kinase activity. The test agent has at least one observable indicator of circadian rhythm function and can be provided to cell preparations, tissues, organs, organisms or animals that express AMPK. The ability of an agent to modulate circadian rhythm can be tested in a variety of animal species that exhibit signs of circadian rhythm function, as well as organs, tissues and cells derived from such animals, and cell preparations derived from them . Agents that modulate AMPK activity can then be identified as agents that have putative circadian rhythm modulating activity.

  A variety of in vitro screening methods are useful for identifying antagonists or agonists that modulate circadian rhythm. The ability of a compound to modulate AMPK is, for example, binding to AMPK to activate or inactivate AMPK, block downstream kinase activity, modulate phosphorylation and dephosphorylation, or by AMPK It can be indicated by the ability of the compound to modulate the predetermined signal produced. Thus, signaling and binding assays can be used to identify antagonists or agonists of AMPK provided in the methods of the present disclosure for identifying compounds that modulate circadian rhythm.

  An “agent” is useful for modulating a protein activity associated with any substance or combination of substances, eg, AMPK activation cascade, that is useful to achieve an objective or result (eg, AMPK Dependent phosphorylation events), or substances or combinations of substances that are useful for modifying or affecting protein-protein interactions or ATP metabolism.

  Exemplary agents include, but are not limited to, members of random peptide libraries (eg, Lam et al., Nature, 354: 82-84, 1991; Houghten et al., Nature, 354). : 84-86, see 1991), including peptides such as soluble peptides, and combinatorial chemistry-derived molecular libraries made with amino acids in the D and / or L configuration, phosphopeptides (limited to them) Not included, but random or partially degenerate members of a directed phosphopeptide library; see, eg, Songang et al., Cell 72: 767-778, 1993), antibodies (to them) Without limitation, polyclonal, monoc Including, final, humanized, anti-idiotype, chimeric or single chain antibodies, and Fab, F (ab ′) 2 and Fab expression library fragments, and epitope binding fragments thereof, organic or inorganic small molecules (eg, So-called natural products or members of chemical combinatorial libraries), molecular complexes (eg protein complexes), or nucleic acids.

  Libraries useful in the methods of the present disclosure (eg, combinatorial chemical libraries) include, but are not limited to, peptide libraries (eg, US Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37, 487-493, 1991; Houghton et al., Nature, 354: 84-88, 1991; see PCT Publication No. WO 91/19735), encoded peptides (eg, PCT Publication). No. WO 93/20242), random bio-oligomers (eg PCT publication WO 92/00091), benzodiazepines (eg US Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides. er) (Hobbs et al., Proc. Natl. Acad. Sci. USA, 90: 6909-6913, 1993), vinylogous polypeptide (Hagihara et al., J. Am. Chem. Soc, 114: 6568). P., 1992), non-peptidic peptidomimetics with glucose scaffolds (Hirschmann et al., J. Am. Chem. Soc, 114: 9217-9218, 1992), similar organic synthesis of small compound libraries ( Chen et al., J. Am. Chem. Soc, 116: 2661, 1994), oligocarbamate (Cho et al., Science, 261: 1303, 1003) and / or peptidylphosphonate (Campbell et al., J Chem., 59: 658, 1994), nucleic acid library (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N. Y., 1989; Ausubel et al., Cul. , Green Publishing Associates and Wiley Interscience, NY, 1989), peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083), antibody libraries (see, eg, Vaughn et al., Nat. Biotechnol, 14: 309-314, 1996; see PCT Application No. PCT / US96 / 10287), carbohydrate libraries (eg Liang et al. Science, 274: 1520-1522, 1996; USA) No. 5,593,853), small organic molecule libraries (eg, benzodiazepines, Baum, C & EN, January 18, 33, 1993; isoprenoids, US Pat. No. 5,569,588; Thiazolidionone and metathiazone, US Pat. No. 5,549,974; pyrrolidine, US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; benzodiazepines, (See 5,288,514) Including the.

  Libraries useful for the disclosed screening methods include, but are not limited to, spatially aligned multipin peptide synthesis (Geysen et al., Proc Natl. Acad. Sci., 81 (13): 3998). ˜4002 (1984), “tea bag” peptide synthesis (Houghten, Proc Natl. Acad. Sci., 82 (15): 5131-5135, 1985), phage display (Scott and Smith, Science, 249: 386-390, 1990), spot or disc synthesis (Ditrich et al., Bioorg. Med. Chem. Lett., 8 (17): 2351-2356, 1998), or on beads Split and Synthetic solid phase synthesis (Furka et al., Int. J. Pept. Protein Res., 37 (6): 487-493, 1991; Lam et al., Chem. Rev., 97 (2): 411 448 (1997)). The library can have various numbers of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members. For example, up to about 500,000 members or even more than 500,000 members.

  In one embodiment, the high-throughput screening method includes providing a combinatorial chemistry or peptide library containing a large number of potential therapeutic compounds (eg, influencing factors of AMPK protein-protein interactions). Such combinatorial libraries are then screened in one or more assays as described herein to display the desired characteristic activity (eg, increase or decrease AMPK protein-protein interaction). ) Identify library members (especially chemical species or subclasses). The compounds thus identified can be used as normal “lead compounds” or can themselves be used as potential or actual therapeutic agents. In some cases, a pool of candidate agents may be identified and further screened to determine which individual agent or a smaller pool of collective agents has the desired activity. Agents that affect (eg, increase or decrease) AMPK interactions or AMP-dependent phosphorylation of processes may have the effect of modulating circadian rhythm (eg, sleep behavior) in a subject and are therefore identified. It is desirable.

  In the screening methods described herein, tissue samples, isolated cells, isolated polypeptides and / or test agents can be presented in a manner suitable for high throughput screening. For example, one or more isolated tissue samples, isolated cells or isolated polypeptides can be placed in a well of a microtiter plate and one or more test agents can be added to the well of the microtiter plate. Alternatively, one or more test agents can be presented in a high-throughput format such as a microtiter plate well (in solution or attached to the plate surface), one or more isolated tissue samples, isolated cells And / or contacting the isolated polypeptide with conditions that at least maintain the tissue sample or isolated cells or desired polypeptide function and / or structure. The test agent may be added to the tissue sample, isolated cell or isolated polypeptide at any concentration that is not lethal to the tissue or cells and does not adversely affect the polypeptide structure and / or function. it can. Different test agents are expected to have different effective concentrations. Thus, in some methods it is advantageous to test a range of analyte concentrations.

  Methods for detecting protein phosphorylation are conventional (eg, Gloffke, The Scientist, 16 (19): 52, 2002; Creaton et al., Cell, 119: 61-74, 2004). See, Detection kits are available from a variety of commercial sources (eg Upstate (Charlottesville, VA, USA), Bio-Rad (Hercules, CA, USA), Marligen Biosciences, Inc. (Ijamsville, MD, USA), Calbiochem (San Diego, CA, USA)). Briefly, phosphorylated protein can be detected using staining specific for the phosphorylated protein in the gel. Alternatively, antibodies specific for phosphorylated proteins can be made or are commercially available. Antibodies specific for phosphoproteins can be inter alia tethered to beads (including beads with a specific color signature) or used in ELISA or Western blot assays.

  In certain methods, the phosphorylation of a polypeptide can be determined when such post-translational modifications are measured detectably, or when such post-translational modifications are detected in a control measurement (eg, the same test system prior to addition of the test agent). Or a similar test system in the absence of the test agent, or a similar test system in the absence of AMPK) at least 20%, at least 30%, at least 50%, at least 100% or at least 250. If the percentage is higher, it is increasing.

  The basic amino acid sequence of an AMPK subunit (eg, AMPKα1 and / or AMPKα2) (and the nucleic acid sequence encoding the basic AMPK subunit (eg, AMPKα1 and / or AMPKα2)) is well known. Exemplary AMPKα1 amino acid sequences and corresponding nucleic acid sequences are, for example, GenBank accession number NM — 206907.3 (GI: 9557298) (Homo sapiens transcript variant 2 REFSEQ including amino acid and nucleic acid sequences); NM — 006251.5 (GI: 94557300) ) (Homo sapiens transcript variant 1 REFSEQ comprising amino acid and nucleic acid sequence); NM_001013367.3 (GI: 9468060) (Mus musculus REFSEQ comprising amino acid and nucleic acid sequence); NMJ) 01039603.1 (GI: 88858444) (amino acid and Gallus gallus REFSEQ containing the nucleic acid sequence; and NM_019142.1 (GI: 11862) Described norvegicus REFSEQ) containing 79XRaJfWS amino acid and nucleic acid sequences. Exemplary AMPKα2 amino acid sequences and corresponding nucleic acid sequences are, for example, GenBank accession number NM_006252.2 (GI: 46887067) (Homo sapiens REFSEQ including amino acid sequence and nucleic acid sequence); NM_178143.1 (GI: 54792085) (amino acid sequence) Mus_culus REFSEQ), including NM_001039605.1 (GI: 8885850) (Gallus gallus REFSEQ including amino acid sequence and nucleic acid sequence); and NM_21466.1 (GI: 47523597) (Mus musculus including amino acid sequence and nucleic acid sequence) REFSEQ).

  In some method embodiments, the homologue or functional variant of the AMPK subunit is at least 60% amino acid sequence identity with the basic AMPKα1 and / or AMPKα2 polypeptide, eg, GenBank accession number NM — 206907.3; NM — 006251.5. NMJ) 01103367.3; NM_001039603.1; NM_019142.1; NM_006252.2; NM_178143.1; NM_001039605.1; or NM_2144266.1, and at least 75%, at least 80%, at least 85% Share at least 90%, at least 95% or at least 98% amino acid sequence identity. In other method embodiments, the homologue or functional variant of the AMPK subunit comprises one or more conservative amino acid substitutions compared to the basic AMPKα1 and / or AMPKα2 polypeptide, eg, GenBank accession number NM_206907.3; NM_006251 NM_001013367.3; NM_001039603.1; NM_019142.1; NM_006252.2; NM_178143.1; NM_001039605.1; or NM_2144266.1, 3 or less, 5 or less, 10 or less , 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, or 50 or less. Exemplary conservative amino acid substitutions have been previously described herein.

  Some method embodiments include a functional fragment of AMPK or a subunit thereof (eg, AMPKα1 and / or AMPKα2). A functional fragment of AMPK or a subunit thereof (eg, AMPKα1 and / or AMPKα2) includes, for example, about 20, about 30, about 40, about 50, about 75, about 100, about 150, or about 200 consecutive amino acid residues Can be any part of the full-length or intact AMPK polypeptide complex or a subunit thereof (eg AMPKα1 and / or AMPKα2), provided that the fragment is at least one AMPK of interest (or AMPKα1 and / or AMPKα2). ) Subject to maintaining function. Protein-protein interactions between polypeptides in the AMPK pathway are thought to include at least the AMPKα subunit (eg, AMPKα1 and / or AMPKα2).

  “Isolated” biological components (eg, polynucleotides, polypeptides or cells) have been purified separately from other biological components in the mixed sample (eg, cell or tissue extracts). For example, an “isolated” polypeptide or polynucleotide is a polypeptide separated from other components of the cell in which the polypeptide or polynucleotide was present (eg, an expression host cell for a recombinant polypeptide or polynucleotide) or It is a polynucleotide.

  The term “purified” refers to the removal of one or more extraneous components from a sample. For example, when a recombinant polypeptide is expressed in a host cell, the polypeptide is purified, for example, by removing host cell proteins, thereby increasing the percentage of recombinant polypeptide in the sample. Similarly, if a recombinant polynucleotide is present in the host cell, the polynucleotide is purified, for example, by removing the host cell polynucleotide, thereby increasing the proportion of the recombinant polynucleotide in the sample.

  An isolated polypeptide or nucleic acid molecule is typically at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 99% (w / w or w / v) of the sample. ).

  Polypeptides and nucleic acid molecules are isolated by methods generally known in the art and described herein. The purity of a polypeptide or nucleic acid molecule may be determined by a number of well-known methods such as polyacrylamide gel electrophoresis for polypeptides or agarose gel electrophoresis for nucleic acid molecules.

  Similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between these sequences. Sequence identity is typically expressed in terms of percent identity. The higher the percentage, the more similar the two sequences.

  Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch, J. et al. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. ScL USA 85: 2444, 1988; Higgins and Sharp, Gene 73: 237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 8: 108. 10890, 1988; Huang et al., Computer Applications in the Biosciences 8: 155-165, 1992; Pearson et al., Methods in Molecular Biology 24: 307-331, 1994; . Lett. 174: 247-250, 1999. Altschul et al. Provide a detailed discussion of sequence alignment methods and homology calculations (J. Mol. Biol. 215: 403-410, 1990). The Basic Local Alignment Search Tool (BLAST ™, Altschul et al., J. Mol. Biol. 215: 403-410, 1990) of the National Center for Biotechnology Information (NCBI) is blastp, blastn, blastx, tblastn and Available from various sources including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet for use in connection with the sequence analysis program of tblastx. A description of how sequence identity is determined using this program is available on the Internet from the help section for BLAST ™.

  For comparisons of amino acid sequences greater than about 30 amino acids, the “Blast2 sequence” function of the BLAST ™ (Blastp) program uses the default parameters (gap start cost [default = 5]; gap extension cost [default = 2]; Mismatch penalty [default = -3]; match reward [default = 1]; expectation value (E) [default = 10.0]; word size [default = 3]; number of entries in one line (V) [default = 100]; using the default BLOSUM62 matrix set to the number of alignments to be displayed (B) [default = 100]). When aligning short peptides (less than about 30 amino acids), alignment is performed using the Blast2 sequence function with the PAM30 matrix set to the default parameters (start gap 9, extension gap 1 penalty). Proteins with even greater similarity to the reference sequence show an increase in percent identity when assessed by this method.

  For comparison of nucleic acid sequences, the “Blast2 sequence” function of the BLAST ™ (Blastn) program is used with the default parameters (gap start cost [default = 11]; gap extension cost [default = 1]; expected value (E) [Default = 10.0]; word size [default = 11]; number of lines described in one line (V) [default = 100]; number of alignments to be displayed (B) [default = 100] This is done using the BLOSUM62 matrix. Nucleic acid sequences with even greater similarity to the reference sequence show an increase in percent identity when assessed by this method.

  Specific binding refers to a specific interaction between one binding partner (eg, a binding agent) and the other binding partner (eg, a target). Such interactions are mediated by one or typically more non-covalent bonds between binding partners (or more often between specific regions or parts of each binding partner). In contrast to non-specific binding sites, specific binding sites can be saturated. Thus, one exemplary method of characterizing specific binding is by a specific binding curve. A specific binding curve shows, for example, the amount of one binding partner bound to a certain amount of one binding partner (first binding partner) as a function of the concentration of the first binding partner. As the concentration of the first binding partner increases under these conditions, the amount of bound first binding partner saturates. In contrast to non-specific binding sites, specific binding partners involved in direct association with each other (eg, protein-protein interactions) can be removed from such associations (eg, protein complexes) from excess amounts. It can be removed (or substituted) competitively by any specific binding partner. Such competition assays (or displacement assays) are well known in the art.

  The present disclosure also provides methods and agents for identifying agents useful for producing circadian rhythm and sleep behavior.

  The following examples are provided to illustrate certain particular features and / or embodiments. These examples are not to be construed as limiting the invention to the particular features or embodiments described.

Example 1
AMPK contributes to metabolic changes in circadian rhythms in fibroblasts. In view of the importance of feeding-derived signals for circadian clock reset, regulation of AMPK by glucose utilization, and accumulated evidence for a role of AMPK in cryptochrome destabilization, AMPK expression and glucose utilization Was examined for circadian rhythm in fibroblasts. When wild-type fibroblasts are cultured in medium containing restricted glucose, the amplitude of circadian reverba and dbp expression is markedly enhanced (Figures 1A and 4), indicating that glucose deficiency causes AMPK. Consistent with a model that activates and reduces CRY stability, which leads to derepression of the CLOCK: BMAL1 targets reverba and dbp. As expected, adding AICAR to the culture medium mimics the effects of glucose deficiency. Remarkably, neither glucose deficiency nor AICAR treatment affected the expression of reverbα and dbp in MEF lacking AMPK (ampkα1 ^{− / −} ; ampkα2 ^{− / −} , “AMPK − / −”) (FIGS. 1A and 4) This indicated that the effect of glucose restriction on fibroblast circadian rhythm was mediated by AMPK.

The Bmal1 promoter is repressed by REVERBα. Therefore, the effect of reduced glucose utilization on circadian rhythm was examined using fibroblasts that stably express luciferase under the control of the Bmal1 promoter. Under standard (high glucose) culture conditions, a high amplitude circadian rhythm of Bmal1-luciferase expression was observed with a period of 25.3 hours (FIGS. 1B, C). A decrease in the amount of glucose in the medium increased the circadian cycle to 30.7 hours. When Bmal1-luciferase expressing cells are cultured in high glucose medium supplemented with AICAR, the circadian cycle is similar to that observed with low glucose and the circadian effects of glucose deficiency are mediated by AMPK. Reinforced the idea that The increased expression of REVERBα observed under restricted glucose conditions is expected to result in decreased expression of genes repressed by REVERBα, including Bmal1. Indeed, activation of AMPK by either a decrease in glucose concentration or AICAR treatment reduced the amplitude of Bmal1-luciferase expression (FIG. 1D).
Together, these results indicate that the circadian rhythm of cultured fibroblasts is responsible for altering glucose utilization and that these effects are mediated by AMPK-induced phosphorylation.

  Regulation of circadian AMPK in vivo. To examine circadian regulation of AMPK, AMPK transcription, localization and substrate phosphorylation were examined in the peripheral organs of intact animals. All experiments were performed with animals kept in constant darkness after synchronization to a standard light-dark cycle, and the observed effect was more than a circadian response to changes in the external environment. Rather it was ensured to be circadian.

  Phosphorylation of both AMPK substrates examined, ACC1-Ser79 and Raptor-Ser792, is more reproducibly higher in the main day than in the night (FIG. 2A), almost corresponding to the daytime when the negative feedback protein is unstable. This was consistent with a model in which rhythmic AMPK activation contributes to degradation. While searching for circadian regulation of AMPK in mouse liver, robust circadian expression of regulatory ampkβ2 subunit with peak expression (FIG. 2C) consistent with minimal nuclear cryptochrome protein time (FIG. 2C) 2B). AMPKβ2 has been reported to drive the nuclear localization of the AMPK complex, whereas the AMPKβ1-containing complex targets the plasma membrane. Thus, circadian transcription of ampkβ2 suggests that fluctuating AMPKβ2 regulates the nuclear localization of AMPKα1 and AMPKα2 in a circadian manner. To test this hypothesis, AMPKα1 and AMPKα2 protein levels in liver nucleus collected throughout the circadian cycle were measured (FIG. 2C), and the rhythmicity of nuclear AMPKα1 peaks in sync with ampkβ2 expression. Was observed. AMPKα2 contained a nuclear localization signal and was always present in the nucleus. The time at which AMPKα1 nuclear localization peaks is also the time of minimal CRY1 protein in the liver nucleus, indicating that rhythmic transfer of AMPK to the nucleus contributes to AMPK-mediated phosphorylation and cryptochrome degradation. Suggest to get.

AMPK changes the circadian clock in vivo. Gene deletion of both AMPKα1 and AMPKα2 in mice leads to early embryonic lethality. Thus, to further investigate the role of AMPK in the liver circadian clock, circadian proteins and transcripts were control mice (LKB1 ^{+ / +} ) or liver bred in constant darkness after synchronization to the light-dark cycle. The cells were examined over 24 hours in the litter livers (LKB1 ^{L / L} ) that had lost lkb1 in the cells. A liver-specific deletion of lkb1 abolishes AMPK activation in this organ and throughout the circadian cycle, especially during the daytime when AMPK was found to be most active in unmodified mice. The amount of CRY1 and CRY2 proteins present in the liver nucleus was significantly increased (FIG. 3B). This increase was associated with a decrease in REVERBα expression during the period corresponding to daylight (FIG. 3B) and a decrease in circadian transcript amplitude throughout the circadian cycle (FIG. 3C). Thus, in vivo loss of AMPK signaling stabilizes cryptochrome, disrupts circadian rhythms, and establishes a synchronization mechanism for the light-independent peripheral circadian clock.

  Although the present disclosure has been described with an emphasis on specific embodiments, it will be apparent to those skilled in the art that variations of the specific embodiments may be used. It is intended that it can be done in other ways than described in. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of this disclosure as defined by the following claims.

Claims (17)

Use of an AMP kinase agonist or antagonist for the manufacture of a medicament for modulating circadian rhythm in a subject. The AMPK agonist is a biguanide derivative, AICAR, metformin or a derivative thereof, phenformin or a derivative thereof, leptin, adiponectin, AICAR (5-aminoimidazole-4-carboxamide, ZMP, DRL-16536, BG800 compound (Betagenon), and furan 2. Use according to claim 1 selected from the group consisting of -2-carboxylic acid derivatives. 2. Use according to claim 1, wherein the subject is a mammal. The use according to claim 1, wherein the effective amount is from about 0.5 mg / kg per day in a single or divided dose to about 100 mg / kg per day. The use according to claim 1, wherein the compound is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial, topical, intraocular, suppository or for subcutaneous injection. A composition comprising an AMP kinase agonist and at least one other circadian rhythm modifying agent. 7. The composition of claim 6, wherein the at least one other circadian rhythm modifying agent is a sleep aid. The AMPK agonist is a biguanide derivative, AICAR, metformin or a derivative thereof, phenformin or a derivative thereof, leptin, adiponectin, AICAR (5-aminoimidazole-4-carboxamide, ZMP, DRL-16536, BG800 compound (Betagenon), and furan The composition of claim 6, which is selected from the group consisting of 2-carboxylic acid derivatives. 7. The composition of claim 6, wherein the compound is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial delivery, topical, intraocular, suppository, or for subcutaneous injection. object.
A method of modulating sleep in a mammal, the method comprising administering to the mammal an amount of an AMPK agonist effective to modulate circadian rhythm in the mammal. The method of claim 10, wherein the mammal is a human. The method according to claim 10, wherein the circadian rhythm is sleep behavior. A method of identifying an agent that modulates circadian rhythm or sleep in a subject comprising:
(A) contacting a sample comprising the AMPK pathway with at least one test agent; and (b) comparing the activity of AMPK or the AMPK pathway in the presence and absence of the test agent. Wherein the test agent that alters the activity is an agent that modulates the activity of circadian rhythm,
Method. Use of a composition according to claim 6, 7 or 8 for modulating circadian rhythm in a subject. 15. Use according to claim 14, wherein the subject is a mammal. 15. Use according to claim 14, wherein the effective amount is from about 0.5 mg / kg per day in a single or divided dose to about 100 mg / kg per day. 15. Use according to claim 14, wherein the compound is formulated for oral administration, intravenous injection, intramuscular injection, epidural delivery, intracranial, topical, intraocular, suppository or for subcutaneous injection.

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